Composition comprising retinol, a precursor or a reaction product of it and a plant extract from at least one Chamomilla plant for the treatment of cancer

ABSTRACT

The invention relates to a (i) Vitamin A, a reaction product, a metabolite or a precursor of it and (ii) a plant extract of chamomile or an active component thereof, preferably for use in the treatment of cancer. The inventive composition may be provided as a medicament or a pharmaceutical composition. The at least one  Chamomilla  plant typically contained in the inventive compositions preferably comprises  Matricaria recutita , more preferably flores tubiformis of  Matricaria recutita . Esters of Retinol typically contained in the inventive compositions typically comprise e.g. Retinyl acetate and a plant extract from the plant  Matricaria recutita.

This application is a continuation of U.S. Ser. No. 13/813,873, filed onFeb. 1, 2013; which is a U.S. national phase of InternationalApplication No. PCT/EP2011/003923, filed Aug. 4, 2011, which claims thebenefit of U.S. Provisional Application Ser. No. 61/370,901, filed Aug.5, 2010, the disclosure of each of which is hereby incorporated byreference in its entirety.

The invention relates to a (i) Vitamin A, a reaction product, ametabolite or a precursor of it and (ii) a plant extract of chamomile oran active component thereof, preferably for use in the treatment ofcancer. The inventive composition may be provided as a medicament or apharmaceutical composition. The at least one Chamomilla plant typicallycontained in the inventive compositions preferably comprises Matricariarecutita, more preferably flores tubiformis of Matricaria recutita.Esters of Retinol typically contained in the inventive compositionstypically comprise e.g. Retinyl acetate and a plant extract from theplant Matricaria recutita.

In the recent years many prior art documents have been published, whichutilize plant extracts for various purposes, e.g. for therapeutic uses,however, only some of these appear to effectively support or even allowan effective treatment.

In this context, WO 03/101479 A1 discloses a composition having avariety of therapeutic and cosmetic uses comprising various components,particularly (i) an antioxidant or a vitamin; (ii) metal ions, and (iii)an agent that enhances the ability of a cell to take up extracellularcomponents. The antioxidants or vitamins may be e.g. any of vitamins C,A and E, the metal ions are typically selected from e.g. monovalent ionssuch as Na, K, or from multivalent ions such as FE, Mo, Mg, Mn, Ca, Zn,Cu or Co, typically in the form of a salt. The agent that enhances theability of a cell to take up extracellular components may be e.g.insulin or a growth hormone or may comprise an antihistamine. Thecomposition of WO 03/101479 A1 is believed to be useful for treatingvarious diseases, e.g., genetic disorders, skin diseases, cancer andviral infections. The composition is typically administered byintramuscular injection. In addition to the composition described in WO03/101479 A1, further active materials may be given to a subject to betreated. Such active materials may comprise steroids such as estradiol,nandrolone or estriol, or vitamins such as Vitamins A, D and/or E.According to WO 03/101479 A1 the function of vitamin A is to preservethe integrity of epithelial tissue, to play a role in protein synthesis,and to stabilize cell membranes and also subcellular membranes. As shownin the therapeutic Examples in WO 03/101479 A1, these active materialsare given separately and do not form part of the composition. E.g.Vitamin A was given separately every week. Furthermore, estriol wasgiven separately every day. Finally, nandrolone decanoate was givenseparately every 20 days. Even though the composition according to WO03/101479 A1 appears to provide some advantageous properties in thetreatment of diseases such as genetic disorders, skin diseases, cancerand viral infections, the composition according to WO 03/101479 A1necessarily requires the presence of insulin in the composition, whichis not only acting as a utilizer of glucose uptake in the cells but alsoacts as a growth hormone but in some cases may also enhance the growthof certain types of cancer. For non-diabetic humans, continued insulinintake, e.g. during cancer therapy, may even lead to hypo-glycemia.Furthermore, the antihistamine present in the composition has a sedativeside-effect. Besides that, preparation of the medicament is relativelycost intensive due to the high number of components.

Furthermore, WO 2008/146009 A1 discloses a composition comprising anaqueous extract of chamomile flowers for the treatment of aproliferative and/or inflammatory condition. Additionally to thechamomile extract the composition may include black cumin seed oil.According to WO 2008/146009 A1, said extracts can reduce DNA synthesisin human cancer cells and inhibit the production of leucotrienes andIL-6 (interleukin 6). It also has been argued that the inhibition ofleucotriene synthesis of the volatile oil is potentiated synergisticallyin the presence of the seed oil of black cumin (Nigella sativa). Moreprecisely, the invention is based on data obtained using an aqueousextract of chamomile flower heads, preferably obtainable by steamdistillation, wherein the aqueous extract is composed of the volatilecomponents of the flower heads of Matricaria recutita L., also known forthose skilled in the art as Matricariae aetheroleum, described in PhEur5.1. The invention is based further on data obtained by using acombination of black cumin seed oil and the volatile oil of thechamomile flower heads. The extracts from WO 2008/146009 A1 alreadyprovide a good basis for therapy.

Another composition utilizing plant extracts is described in WO2009/138860 A1. It discloses a composition comprising an aqueous and/ororganic extract of at least one Chamomilla plant and/or of at least oneAchillea plant for the treatment of an abnormal proliferative and/orviral condition, with the proviso that the mono/single extract ofAchillea millefolium (L.) is excluded from the treatment of saidabnormal proliferative condition, and with the further proviso that themono/single extract of Matricaria chamomilla (L.) is excluded from thetreatment of said viral condition.

However, none of these prior art documents provides or suggests acomposition, which allows to effectively treat cancer, either as asupplementary therapy or as a single therapy. In this context,particularly solid tumours of neurogenic, mesenchymal or epithelialorigin and metastases thereof represent a major obstacle.

Accordingly, it is an object of the present invention to provide acomposition for the treatment of cancer, preferably solid tumours ofneurogenic, mesenchymal or epithelial origin and metastases thereof. Itis furthermore an object of the present invention to provide amedicament containing the composition for the treatment of cancer.Preferably, the composition should consist only of compounds essentialfor the desired activity profile. The composition should also worksynergistically, inhibit tumour cell proliferation and induce at leastcell death of cancer cells.

The described objectives are solved by the present invention, preferablyby the subject matter of the attached claims. More preferably, thepresent invention is solved according to a first embodiment by acomposition comprising or consisting of (i) Vitamin A, a reactionproduct, a metabolite or a precursor of it and (ii) a plant extract ofchamomile or an active component thereof, preferably for use in theprevention, treatment or amelioration of cancer or tumour disease. Insome embodiments of the present invention the compositions according tothe present invention do not comprise—aside of the plant extract ofchamomile—further plant extracts and/or do not comprise plant oil asactive ingredient. Most preferably, the inventive composition consistsof components (i) and (ii) or contains components (i) and (ii) as theonly (pharmaceutically) active ingredients of the inventive composition.

The present invention is based on the surprising finding of the presentinventors that a composition comprising (i) Retinol and derivativesthereof and plant extracts of Matricaria recutita efficiently inhibitsin a synergistic way tumour cell proliferation and induces cell death ina number of tumour cell lines at doses, which have not been shown to beeffective to inhibit tumour cell proliferation and induce cell death,when given alone. More surprisingly it was found by the presentinventors that Retinol and Retinoic acid, when administered inphysiological concentrations, induced together with plant extracts ofMatricaria recutita, cell death in tumour cells derived from neurogenicand epithelial tumours. The present invention is based on data obtainedusing an aqueous extract of Matricaria recutita, including water steamdistillate of Matricaria recutita, and on data using an organic extract,especially alcoholic extracts of the tubular flowers of Matricariarecutita L., (Flores tubiformis) in combination with an Ester of Retinol(Retinylacetat) or Retinoic acid. The experiments were performed usingthe human glioblastoma cell lines A172 and U87 and the human large celllung cancer cell line H460. When incubated with extracts of chamomile,the tumour cells were affected similar to the studies described in WO2009/138860, while incubated with the inventive composition, a strongincrease with respect to the inhibition of tumour cell proliferation andinduction of cell death took place, which was not observed in theexamples described in WO 2009/138860. Therefore treatment of cancer withchamomile extracts involving retinoids might help generally and incertain sub types of cancer.

The inventive composition may be prepared by admixing the components ofthe inventive composition, preferably comprising or consisting of (i)Vitamin A, a reaction product, a metabolite or a precursor of it and(ii) a plant extract of chamomile or an active component thereof. Inthis context, the inventive composition preferably comprises about 50 mgto about 1500 mg, preferably about 50 mg to about 1000 mg, of anchamomile extract as described above, and/or typically about 3 mg toabout 90 mg, preferably about 3 mg to about 90 mg Vitamin A, or any ofthe Vitamin A reaction products, metabolites or precursors as describedherein.

According to the first embodiment of the present invention, theinventive composition comprises as a first ingredient (i) Vitamin A, areaction product, a metabolite or a precursor of it. In this context,Vitamin A is known to be important for normal vision, gene expression,reproduction, embryonic development, growth, and immune function. It isa fat-soluble vitamin that is essential for humans and othervertebrates. Vitamin A comprises of a family of molecules containing a20 carbon structure with a methyl substituted cyclohexenyl ring(beta-iononic ring) and a tetraene side chain with a hydroxyl group(retinol), aldehyde group (retinal), carboxylic acid group (retinoicacid), or ester group (retinyl ester) at carbon-15. In foods of animalorigin, the major form of vitamin A is an ester, primarily retinylpalmitate, which is converted to an alcohol (retinol) in the smallintestine. The retinol form functions as storage form of the vitamin,and can be converted to and from its visually active aldehyde form,retinal. The associated acid (retinoic acid), a metabolite that can beirreversibly synthesized from vitamin A, has only partial vitamin Aactivity, and does not function in the retina or some essential parts ofthe reproductive system. The term vitamin A also includes precursors ofvitamin A, such as provitamin A carotenoids that are dietary precursorsof retinal. The term retinoids refers to retinol, its metabolites, andsynthetic analogues that have a similar structure. The liver, lung,adipose and other tissues possess carotene 15, 15′-dioxygenase activityand it is presumed that carotenes may be converted to vitamin A as theyare delivered to tissues. The major end products of the enzyme'sactivity are retinol and retinoic acid. Vitamin A deficiency has beenassociated with a reduction in lymphocytic numbers, natural killer cellsand antigen-specific immunoglobulin responses. A decrease in leukocytesand lymphoid organ weights, impaired T cell function, and decreasedresistance to immunologic tumours have been observed with inadequatevitamin A intake. A generalized dysfunction of humoral and cell-mediatedimmunity is common in experimental animals and is likely to exist inhumans (see e.g. “Reference Intakes for Vitamin A. Food and NutritionBoard, Institute of Medicine, 2001”). Accordingly, in the context of thepresent invention, Vitamin A, a reaction product, a precursor or ametabolite there from, typically comprises as Vitamin A or a derivativethereof, preferably e.g. retinoic acid, all-trans-retinoic acid,13-cis-retinoic acid, esters of Vitamin A, including retinyl palmitate,or retinol, its visually active aldehyde form retinal, retinyl acetate,alitretinoin, as a precursor of Vitamin A preferably beta-carotene,alpha-carotene, gamma-carotene or beta-cryptoxanthine and as metaboliteof Vitamin A preferably the associated acid (retinoic acid), ametabolite that can be irreversibly synthesized from vitamin A, has onlypartial vitamin A activity.

It is to be noted that the prior art also discloses an example of theuse of a retinoid in cancer treatment in acute promyelocytic leukemia(APL) in supraphysiological concentrations (see Wang, Z. Y., and Chen,Z. (2008) Blood 111, 2505-2515). Acute promyelocytic leukemia (APL) is adistinct subtype of acute myeloid leukemia (AML) and is unique fromother forms of AML because it responses exceptionally well to all-transretinoic acid (ATRA). In APL patients, the differentiation program ofpromyleocytes is halted due to a mutated retinoic acid receptor alpha(RAR-alpha). The mutation is caused by a chromosomal translocationbetween the promyelocytic leukemia gene (PML) on chromosome 15 and theRAR-alpha gene on chromosome 17 resulting in a fusion protein PML-RAR.This hybrid protein represses gene expression of target genes that areessential for the cell differentiation to granulocytes by binding to aset of co-repressors (CoR) and response elements (RAREs). Much of thetherapeutic effect of ATRA appears to be derived from its ability tocause a configuration change of the fusion protein leading to, firstly,dissociation of the CoRs and RAREs and, secondly, increase affinity tocoactivator complexes which enables, once again, the transcription ofgenes necessary for differentiation. In addition, ATRA is also thoughtto induce ubiquitylation and proteasome-mediated degradation of thePML-RAR fusion protein, resulting in relieving the block todifferentiation in APL.

The inventive composition may furthermore comprise according to thefirst embodiment (ii) a plant extract of chamomile or of an activecomponent thereof.

Such a plant extract of chamomile or an active component thereof istypically derived from at least one chamomilla plant, preferably fromMatricaria recutita or German chamomile. The plant extract of chamomileor an active component thereof is typically derived from the wholeplant, more preferably from the flowerheads, even more preferably fromtubular flowers of the plant, and likely more preferably from tubularflowers of Matricaria recutita L. and most preferably tubular flowersfrom the inflorescence of Matricaria recutita L. In this context,Matricaria recutita or German chamomile, also spelled chamomile, is anannual plant of the composite family Asteraceae. Synonyms are:Chamomilla chamomilla, Chamomilla recutita (as defined according to theFlora Europaea or as described in the European Pharmacopeia (Matricariaeaetheroleum PhEur 5, corrected. 5.1). Matricaria chamomilla, andMatricaria suaveolens. Notably, the flower head (capitulum) of thechamomile plant is usually composed of two parts, i.e. the yellowdisc-shaped or tubular flowers or florets (flores tubiformis ortubiflorum) and the white radiating flowers or florets (floresligutatea). It has been found that organic extracts of the tubularflowers of Matricaria recutita L. (Flores tubiformis) are suitable forthe synchronization and the S-phase arrest of abnormal proliferativemammalian cells, especially of cancer cells. This synchronization takesplace due to the induction of ornithine decarboxylase (transfer fromG₀-phase into G₁-phase) and the inhibition of topoisomerase II(accumulation and arrest in the early S-phase). It was also found thatthe inhibition of topoisomerase II was more than 100-fold stronger withan organic extract than with an aqueous extract (with respect to theconcentration for complete inhibition of the enzyme). Due to the factthat the inhibition of the topoisomerase II is crucial for theeffectiveness of cell synchronization the inventive compositioncomprising organic extracts of the tubular flowers of Matricariarecutita L. (Flores tubiformis) of the present invention together withVitamin A, a reaction product or a metabolite thereof or a precursorthereof, are much more potent than the corresponding aqueous extracts.

Even though at least one chamomilla plant may be used as a basis for theplant extract of chamomile contained in the inventive composition, plantextracts other than from Matricaria recutita or German chamomile arepreferably excluded from the scope of the present invention by way ofdisclaimer.

The plant extract of chamomile or of an active component thereof may bean aqueous and/or organic extract and may be obtained by any suitableprocedure or method known to those of ordinary skill in the art. Theextract may be obtained by e.g. using an aqueous or organic medium, andseparation of the extract from other components, e.g. by filtration,chromatography, supercritical fluid extraction, steam distillation,preferably by methods or procedures known to those of ordinary skill inthe art. A preferred procedure for the preparation of an aqueous extractis described in WO 2007/057651 A1, the specific disclosure of which isenclosed herein in its entirety. Preferably, such an extract comprises amulti-component mixture of water-soluble components.

Typically, the plant extract from chamomile may be obtained by addingwater to the appropriate plant part to obtain a suspension. Thesuspension is then usually heated to a temperature below the boilingpoint of water, e.g. 90-94° C., and then cooled to room temperature. Theaqueous extract is then preferably subjected to one or two filtrationsteps, preferably as described herein.

Preferably, the plant extract of chamomile or of an active componentthereof or the inventive composition may be prepared as an ethanolicliquid extract or as a waterfree ethanolic liquid extract. For anethanolic liquid extract the chamomile or parts thereof, preferablytubular flowers from chamomile, most preferably tubular flowers from theinflorescence of Matricaria recutita L, are extracted with ethanol,preferably ethanol absolute, preferably under stirring. The obtainedmixture is then preferably stirred, e.g. for about ½ to 2 hours,preferably for about one hour, e.g. at a temperature of about 30° C. to50° C., e.g. about 40° C. The preparation is then preferably filtratedto obtain a liquid extract typically with a solid content of less than2%. A water-free ethanolic liquid extract may be then prepared bymethods known to a person of skill in the art for reducing the watercontent in an organic ethanol containing solution, e.g. by adding to theethanolic liquid extract Span® 20 (Sigma®) (sorbitan (mono)laurate) andTranscutol® HP (Gattefossé®) (diethylene glycol monoethyl ether), e.g.corresponding to a ratio of 1/2/0.2, and evaporating the ethanol underreduced pressure (e.g. 100 to 25 mbar) and a bath temperature of 30° C.to 50° C., e.g. 40° C. Additionally, the extract may be filtered, ifnecessary, e.g. a cellulose filter (e.g. TS 4 Filtrox®). Lutrol L44(BASF) can also be utilized.

Alternatively, the plant extract of chamomile or of an active componentthereof or the inventive composition may be prepared as an aqueousextract. For an aqueous extract the chamomile or parts thereof,preferably tubular flowers from chamomile, most preferably tubularflowers from the inflorescence of Matricaria recutita L., are extractedwith water, preferably aqua dest. or aqua purificata, preferably understirring. The mixture is typically heated up to a temperature of about70° C. to 100° C., more preferably to a temperature of about 90° C. to95° C., preferably in a specific time frame, e.g. 15 minutes to about 2hours, e.g. about 30 minutes to about 35 minutes. Subsequent the mixturemay be cooled down to about 20 to about 40° C., e.g. about 30° C. toabout 35° C., may be filtered once or twice or more, e.g. using a deeplayer cellulose filter. If necessary, the extract may be treated with 1%m/m of Mygliol 812 N, preferably stirring the extract after addition.Furthermore, the pH of the mixture may be adapted to e.g. about 6 to 7,e.g. to about 6.3 to about 6.7, e.g. with a base, e.g. sodium hydroxide,and optionally micro-filtered, e.g. through a 0.1 μm membrane filter andoptionally subsequently through a 1000 kDa ultra filter.

The inventive composition but also the plant extract of chamomile to beused should be suitable for administration, e.g. for oral use, forinjection, etc. For this purpose, it is desirable to remove endotoxins,polyphenols, cumarines and (by any suitable means, known to those in theart) large molecular weight component, e.g. those having a molecularweight of more than 1,000 or 10,000 Da.

As described above the aqueous extract after preparation may besubjected to one or two filtration steps, preferably as describedherein. Such a filtration step may be selected from filtrationtechniques such as, for the purposes of illustration only,microfiltration and/or ultrafiltration, respectively. Other techniques,such as use of a lipophilic barrier, also may be suitable. Eachfiltration step may be conducted in one, two or more than two stages, ifdesired, combining the same of different filtration techniques. In thiscontext, microfiltration is typically applied in order to removematerial that would otherwise compromise the effectiveness of theultrafiltration step.

Furthermore, the inventive composition and/or the plant extract ofchamomile prior to preparing the inventive composition may be purified.A preferred procedure is to purify the initially obtained plant extract,e.g. a volatile oil, an aqueous or organic extract, preferably prior topreparing the inventive composition and/or the inventive compositionsubsequent to preparation, by contacting it with crosspovidone(crosslinked povidone) and sodiumsulfate. Crosspovidone is known forthose skilled in the art to complex phenolic compounds and cumarines.Sodiumsulfate is known to bind residues of water. Separation of thepurifying agents results in a plant extract or composition free ornearly free from cumarine, phenol and residual water. After purificationthe analysis of bacterial endotoxins of the samples obtained may beperformed with the Cambrex PyroGene assay, preferably using a dilutionfactor of 1:10.000.

According to a further embodiment, the present invention provides apharmaceutical composition comprising the inventive composition asdefined herein, preferably comprising or consisting of (i) Vitamin A, areaction product, a metabolite or a precursor of it and (ii) a plantextract of chamomile or an active component thereof, preferably for usein the treatment of cancer, and optionally a pharmaceutically acceptablecarrier and/or vehicle.

The inventive pharmaceutical composition typically comprises a “safe andeffective amount” of the active ingredient(s) of the inventivepharmaceutical composition, particularly of the (i) Vitamin A, areaction product, a metabolite or a precursor thereof, and (ii) a plantextract of chamomile or an active component thereof. As used herein, a“safe and effective amount” means an amount of the active ingredient(s)as defined herein as such that is sufficient to significantly induce apositive modification of a disease or disorder as defined herein. At thesame time, however, a “safe and effective amount” is small enough toavoid serious side-effects and to permit a sensible relationship betweenadvantage and risk. The determination of these limits typically lieswithin the scope of sensible medical judgment. A “safe and effectiveamount” of the active ingredient(s) of the inventive pharmaceuticalcomposition, particularly of the inventive nucleic acid as definedherein, will furthermore vary in connection with the particularcondition to be treated and also with the age and physical condition ofthe patient to be treated, the body weight, general health, sex, diet,time of administration, rate of excretion, drug combination, theactivity of the inventive nucleic acid as defined herein, the severityof the condition, the duration of the treatment, the nature of theaccompanying therapy, of the particular pharmaceutically acceptablecarrier used, and similar factors, within the knowledge and experienceof the accompanying doctor. The inventive pharmaceutical composition maybe used for human and also for veterinary medical purposes, preferablyfor human medical purposes.

The effective dose of the inventive pharmaceutical composition,preferably incorporated in a suitable dosage form as described herein,is thus typically dependent of the type of cancer and the Vitamin AStatus of the patient. However a typical content of an effective dose ofthe inventive pharmaceutical composition, e.g. in form of a capsule,typically comprises about 50 mg to about 1500 mg, preferably about 50 mgto about 1000 mg, of chamomile extract, and/or typically about 3 mg toabout 90 mg, preferably about 3 mg to about 90 mg Vitamin A, for exampleRetinoic acid or any of the Vitamin A precursors, derivatives ormetabolites as described herein. The effective dose, in a form asdescribed herein, could be given once a week, once every two or three orfour or five or six days, one, two, three or four times daily, etc.

Pharmaceutical compositions as defined herein typically can beformulated by methods known to those skilled in the art preferablyutilizing pharmaceutically acceptable components. The term“pharmaceutically acceptable” refers to those properties and/orsubstances which are acceptable to the patient from apharmacological/toxicological point of view and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingfactors such as formulation, stability, patient acceptance andbioavailability.

In this context, a pharmaceutically acceptable carrier and/or vehicletypically includes the liquid or non-liquid basis of the inventivepharmaceutical composition. If the inventive pharmaceutical compositionis to be provided in liquid form, the carrier will be typicallypyrogen-free water; isotonic saline or buffered (aqueous) solutions,e.g. phosphate, citrate etc. buffered solutions. The injection buffermay be hypertonic, isotonic or hypotonic with reference to the specificreference medium, i.e. the buffer may have a higher, identical or lowersalt content with reference to the specific reference medium, whereinpreferably such concentrations of the afore mentioned salts may be used,which do not lead to damage of cells due to osmosis or otherconcentration effects. Reference media are e.g. liquids occurring in “invivo” methods, such as blood, lymph, cytosolic liquids, or other bodyliquids, or e.g. liquids, which may be used as reference media in “invitro” methods, such as common buffers or liquids. Such common buffersor liquids are known to a skilled person. Ringer-Lactate solution isparticularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well for the inventivepharmaceutical composition, which are suitable for administration to apatient to be treated. The term “compatible” as used here means thatthese constituents of the inventive pharmaceutical composition arecapable of being mixed with the inventive nucleic acid as defined hereinin such a manner that no interaction occurs which would substantiallyreduce the pharmaceutical effectiveness of the inventive pharmaceuticalcomposition under typical use conditions. Pharmaceutically acceptablecarriers, excipients and solvents must, of course, have sufficientlyhigh purity and sufficiently low toxicity to make them suitable foradministration to a person to be treated. Some examples of compoundswhich can be used as pharmaceutically acceptable carriers, fillers orconstituents thereof are sugars, such as, for example, lactose, glucoseand sucrose; starches, such as, for example, corn starch or potatostarch; cellulose and its derivatives, such as, for example, sodiumcarboxymethylcellulose, ethylcellulose, cellulose acetate; powderedtragacanth; malt; gelatin; tallow; solid glidants, such as, for example,stearic acid, magnesium stearate; calcium sulfate; vegetable oils, suchas, for example, groundnut oil, cottonseed oil, sesame oil, olive oil,corn oil and oil from theobroma; polyols, such as, for example,polypropylene glycol, glycerol, sorbitol, mannitol and polyethyleneglycol; alginic acid.

Administration of the inventive pharmaceutical composition preferablyoccurs via the respiratory tract, via oral administration or may beadministered by injection, typically via parenteral injection,preferably by subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional,intracranial, transdermal, e.g. via transdermal therapeutic deliverysystems such as dermal patches, intradermal, intrapulmonal,intraperitoneal, intracardial, intraarterial, and sublingual injectionor via infusion techniques. More preferably, administration of theinventive pharmaceutical composition occurs by intravenous or, morepreferably, intramuscular injection, yet most preferably by an inhalatoras an aerosol or micro/nano-emulsion via the respiratory tract or viaoral administration. The composition might also be administered via thegastrointestinal system, e.g. rectally, e.g. via suppositories.

The pharmaceutical composition might be formulated into sterileinjectable forms, into oral dosage forms, into tablets, like filmtablets, or capsules like soft gelatin capsules or hard capsules, forexample hydroxypropylmethylcellulose capsules, troches, lozenges,aqueous or oily suspensions or solutions, dispersible powders orgranules, emulsions, syrups or elixirs, suppositories, etc., any ofthese preferably together with suitable pharmaceutically acceptablecarriers and/or auxiliaries/excipients. If desired the pharmaceuticalcomposition might be incorporated in sustained release formulations,e.g. retard tablets, or any kind of dosage forms suitable to revealoptimal bioavailability, solubility and stability of the pharmaceuticalcomposition.

In the above context, sterile injectable forms/preparations of theinventive pharmaceutical compositions may be e.g. (aqueous oroleaginous) suspensions. These suspensions may be formulated accordingto techniques known in the art using suitable dispersing or wettingagents and suspending agents. The sterile injectable form/preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the pharmaceutically acceptable carriers that maybe employed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents that are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation of the inventive pharmaceutical composition.

The inventive pharmaceutical composition containing the activeingredient(s) may also be preferably in a oral dosage form suitable fororal administration, for example, as capsules, tablets, troches,lozenges, aqueous, or oily suspensions or solutions, dispersible powdersor granules, emulsions, hard or soft capsules, or syrups or elixirs.Such compositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavouring agents, colouring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. In the case of tablets for oral use, carrierscommonly used include lactose and corn starch. Lubricating agents, suchas magnesium stearate, are also typically added. For oral administrationin a capsule form, useful diluents include lactose and dried cornstarch.When aqueous suspensions are required for oral use, the activeingredient, i.e. the inventive nucleic acid as defined herein, iscombined with emulsifying and suspending agents. If desired, certainsweetening, flavouring or colouring agents may also be added. Tabletstypically contain the active ingredient(s) in admixture with non-toxicpharmaceutically acceptable excipients such as, for example, inertdiluents such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example corn starch or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated, to form osmotic therapeutic tablets for controlled release.Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient(s) is mixed with an inert solid diluent,for example calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions in the above context, preferably for oraladministrations, for administrations via the respiratory or thegastrointestinal tract or for injectable forms comprising the inventivepharmaceutical compositions, may contain the active ingredient(s) inadmixture with suitable excipients. Such excipients are suspendingagents, for example sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethyl-cellulose, sodium alginate, polyvinyl pyrrolidone,gum tragacanth and gum acacia; dispersing or wetting agents, for examplea naturally occurring phosphatide such as lecithin, or condensationproducts of an alkylene oxide with fatty acids, for examplepolyoxyethylene stearate, or condensation products of ethylene oxidewith long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol, such as apolyoxyethylene with partial esters derived from fatty acids and hexitolanhydrides, for example polyoxyethylene sorbitan monooleate. Aqueoussuspensions may also contain one or more preservatives, for exampleethyl or n-propyl p-hydroxybenzoate, one or more colouring agents, oneor more flavouring agents, and one or more sweetening agents, such assucrose or saccharin.

Oily suspensions in the above context, preferably for administrationsvia the respiratory or the gastrointestinal tract comprising theinventive pharmaceutical compositions, may be formulated by suspendingthe active ingredient in a vegetable oil, for example arachis oil, oliveoil, sesame oil or coconut oil, or in a mineral oil such as liquidparaffin. The oily suspensions may contain a thickening agent, forexample beeswax, hard paraffin or cetyl alcohol. Sweetening agents (suchas those set forth above) and flavouring agents may be added to providea palatable oral preparation. These compositions may be preserved by theaddition of an anti-oxidant such as ascorbic acid.

Furthermore, dispersible powders and granules suitable for preparationof an aqueous suspension by the addition of water, preferably foradministrations via the respiratory or the gastrointestinal tractcomprising the inventive pharmaceutical compositions, provide the activeingredient(s) in admixture with a dispersing or wetting agent,suspending agent and one or more preservatives. Suitable dispersing orwetting agents and suspending agents are exemplified above. Sweetening,flavouring and colouring agents may also be present. A pharmaceuticalcomposition for use in the invention may also be in the form of anoil-in-water emulsion. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally occurring gums, for example gum acacia or gum tragacanth,naturally occurring phosphatides, for example soya bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate and condensation products ofthe said partial esters with ethylene oxide, for example polyoxyethylenesorbitan monooleate. The emulsions may also contain sweetening andflavouring agents. Syrups and elixirs may be formulated with sweeteningagents, for example glycerol, propylene glycol, sorbitol or sucrose.Such formulations may also contain a demulcent, a preservative andflavouring and colouring agents. The pharmaceutical compositions may bein the form of a sterile injectable aqueous or oleaginous suspension.This suspension may be formulated using suitable dispersing or wettingagents and suspending agents, examples of which have been mentionedabove. A sterile injectable preparation may also be in a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables. Thecomposition may also be administered in the form of suppositories forrectal administration of the drug. Such compositions can be prepared bymixing the drug with a suitable non-irritating excipient which is solidat ordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Such materials arecocoa butter and polyethylene glycols.

Additionally, the inventive pharmaceutical composition may beadministered topically, where applicable. Topical administration isparticularly preferred, when the target of treatment includes areas ororgans readily accessible by topical application, e.g. includingdiseases of the skin or of any other accessible epithelial tissue.Suitable topical formulations are readily prepared for each of theseareas or organs. For topical applications, the inventive pharmaceuticalcomposition may be provided typically in the form of, for example,creams, ointments, jellies, solutions or suspensions, preferablycontaining the active ingredient(s) as defined herein suspended ordissolved in one or more carriers. Carriers for topical administrationinclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, the inventivepharmaceutical composition can be formulated in a suitable lotion orcream. In the context of the present invention, suitable carriersinclude, but are not limited to, mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water.

The inventive pharmaceutical composition may additionally comprise apenetration enhancer. Such a penetration enhancer may be selected fromany compound suitable for the inventive purpose, e.g. selected frompenetration enhancers (also called sorption promoters or accelerants)known to a person of skill in the art, preferably a compound whichpenetrates into skin to reversibly decrease the barrier resistance.Numerous compounds have been evaluated for penetration enhancingactivity, including sulphoxides (such as dimethylsulphoxide, DMSO),Azones (e.g. laurocapram), pyrrolidones (for example 2-pyrrolidone, 2P),alcohols and alkanols (ethanol, or decanol), glycols (for examplepropylene glycol, PG, a common excipient in topically applied dosageforms, diethylene glycol monoethyl ether (Transcutol®)), surfactants(also common in dosage forms) and terpenes, etc. Many potential sitesand modes of action have been identified for skin penetration enhancers;the intercellular lipid matrix in which the accelerants may disrupt thepacking motif, the intracellular keratin domains or through increasingdrug partitioning into the tissue by acting as a solvent for thepermeant within the membrane. Further potential mechanisms of action,for example with the enhancers acting on desmosomal connections betweencorneocytes or altering metabolic activity within the skin, or exertingan influence on the thermodynamic activity/solubility of the drug in itsvehicle are also feasible, and form part of the present disclosure. Eventhough insulin may be included as a penetration enhancer, it ispreferably excluded from the scope of the present invention by way ofdisclaimer.

The inventive composition or pharmaceutical composition may be used toprevent, treat or ameliorate a disease as defined herein, preferablycancer or a tumour disease, either as a supplementary therapy or as asingle therapy. For this purpose, the inventive composition orpharmaceutical composition may be administered via any of the aboveroutes using any of the above defined dosage forms. Furthermore, theinventive composition or pharmaceutical composition may be administeredprior, concurrent and/or subsequent to a (conventional) cancer therapy,e.g. a chemotherapy, a surgery, an immunotherapy, etc.

In this context, cancer or a tumour disease preferably includes e.g.solid tumours of neurogenic, mesenchymal or epithelial origin andmetastases thereof, colon carcinomas, melanomas, renal carcinomas,lymphomas, acute myeloid leukaemia (AML), acute lymphoid leukaemia(ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia(CLL), gastrointestinal tumours, pulmonary carcinomas, gliomas, thyroidtumours, mammary carcinomas, prostate tumours, hepatomas, variousvirus-induced tumours such as, for example, papilloma virus-inducedcarcinomas (e.g. cervical carcinoma), adenocarcinomas, herpesvirus-induced tumours (e.g. Burkitt's lymphoma, EBV-induced B-celllymphoma), heptatitis B-induced tumours (hepatocell carcinoma), HTLV-1-and HTLV-2-induced lymphomas, acoustic neuromas/neurinomas, cervicalcancer, lung cancer, pharyngeal cancer, anal carcinomas, glioblastomas,lymphomas, rectal carcinomas, astrocytomas, brain tumours, stomachcancer, retinoblastomas, basaliomas, brain metastases, medulloblastomas,vaginal cancer, pancreatic cancer, testicular cancer, melanomas,thyroidal carcinomas, bladder cancer, Hodgkin's syndrome, meningiomas,Schneeberger disease, bronchial carcinomas, hypophysis tumour, Mycosisfungoides, oesophageal cancer, breast cancer, carcinoids, neurinomas,spinaliomas, Burkitt's lymphomas, laryngeal cancer, renal cancer,thymomas, corpus carcinomas, bone cancer, osteosarcomas, non-Hodgkin'slymphomas, urethral cancer, CUP syndrome, head/neck tumours,oligodendrogliomas, vulval cancer, intestinal cancer, colon carcinomas,oesophageal carcinomas, wart involvement, tumours of the smallintestine, craniopharyngeomas, ovarian carcinomas, soft tissuetumours/sarcomas, ovarian cancer, liver cancer, pancreatic carcinomas,cervical carcinomas, endometrial carcinomas, liver metastases, penilecancer, tongue cancer, gall bladder cancer, leukaemia, plasmocytomas,uterine cancer, lid tumour, prostate cancer, etc., particularly solidtumours of neurogenic, mesenchymal or epithelial origin and metastasesthereof.

According to a further preferred embodiment, the inventive compositionas defined above, preferably comprising or consisting of (i) Vitamin A,a reaction product, a metabolite or a precursor of it and (ii) a plantextract of chamomile or an active component thereof, may be provided asa pharmaceutical composition as defined above, e.g. in the form of a kitor kit of parts. Such a kit or kit of parts may comprise the inventivecomposition or the inventive pharmaceutical composition either in onepart of the kit or in one or more parts of the kit, e.g. one part of thekit comprising Vitamin A, a reaction product, a metabolite or aprecursor of it, a further part of the kit comprising a plant extract ofchamomile or an active component thereof, and at least one further partof the kit comprising The kit or kit of parts may furthermore containtechnical instructions with information on the administration and dosageof the inventive composition or its components, the inventivepharmaceutical composition or of any of its components or parts, e.g. ifthe kit is prepared as a kit of parts.

In the present invention, if not otherwise indicated, different featuresof alternatives and embodiments may be combined with each other.Furthermore, the term “comprising” shall not be construed as meaning“consisting of”, if not specifically mentioned. However, in the contextof the present invention, term “comprising” may be substituted with theterm “consisting of”, where applicable.

FIGURES

The following Figures are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

FIG. 1: shows the potential growth inhibitory effects of retinyl acetate(1 μg/ml), MR15E7 (ethanolic liquid extract) (30 μg/ml) and thecombination of 30 μg/ml of the extract and retinyl acetate (1 μg/ml)observed on both the A172 (A) and U87 MG (B) cell lines. The solventcontrol and the final ethanol concentration for every well was 2%.

-   -   Results: It appeared that retinyl acetate at 1 μg/ml did not        affect cellular growth in A172 and U87 MG glioblastoma cells as        indicated in FIGS. 1A and 1B. The data derived from the        experiments described in Example 10, however, showed that MR15E7        reduced A172's cell numbers by 30% to 70% as compared to solvent        control (100%). The addition of 1 μg/ml of retinyl acetate        further reduced the cellular growth to 14%, demonstrating a        clear efficacy enhancement of the combination compared to the        compounds alone (FIG. 1A). The effect is also seen similar in        the U87 MG (FIG. 1B) but not as strong as observed for the A172.

FIG. 2: illustrates the effects of retinoic acid (1 μg/ml), MR15E14(ethanolic liquid extract) (21 μg/ml), CHARE (aqueous liquid extract)(21 μg/ml) and the combination of the extracts and retinoic acid (1μg/ml) observed on the H460 cells. The solvent control and the finalethanol concentration for every well was 2%.

-   -   Results: As can be seen 1 μg/ml of Retinoic acid, 21 μg/ml of        MR15E14 and 21 μg/ml of CHARE slightly reduced the cellular        growth of H460 when the cells were treated with the individuals.        However, when the same concentration of Retinoic acid was given        simultaneously with 21 μg/ml of MR15E14 or CHARE, the        anti-proliferative effect was strongly enhanced.

FIG. 3: shows

-   -   (A) the treatment of H460 with MR15E14 (ethanolic liquid        extract) (21 μg/ml), 1% AMT(M) and 1% AMT(UPW) on H460 in        combination with retinoic acid (1 μg/ml) or vehicle control (1%        Ethanol).    -   (B) the treatment of H460 with CHARE (aqueous liquid extract)        (21 μg/ml), 1% AMT(CHARE) and 1% AMT(UPW) on H460 in combination        with retinoic acid (1 μg/ml) or vehicle control (1% Ethanol).        H460 cells were incubation with the sample pairs for a total of        6 days with one dose and media refreshment.    -   Results: As already earlier seen in FIG. 2, retinoic acid itself        showed a slight growth retarding effect on H460 large cell lung        cancer cells and again the results indicate a synergistic effect        between the chamomile extracts and retinoic acid in reducing        cellular growth of H460. The addition of the AMT components to        the chamomile extracts did not enhance this effect (FIG. 3A)        and, even worse, addition of AMT components promoted cell        proliferation in some cases (FIG. 3B). When the components of        AMT were tested alone (without Chamomile extracts) they markedly        increased cell numbers by more than 50% (FIGS. 3A and 3B). AMT        containing the aqueous extract according Example 4 (CHARE) also        caused an increase in cell growth. This was not seen with the        ethanolic extract according Example 2 (MR15E14), probably as the        antiproliferative activity is already alone somewhat stronger        than seen for the aqueous extract. Addition of retinoic acid        abolished the growth promoting effect of AMT (UPW) and as also        seen in the previous pharmacological Examples 10 and 11 enhanced        the anti-proliferative effects of both chamomile extracts. The        results demonstrate the advantages of the use of a combination        solely composed of chamomile and retinoic acid and the        disadvantage of the complex mixture of compounds found in AMT.

FIG. 4: shows the treatment of H69 cells with Blue Chamomile oil (indifferent concentrations) in combination with retinoic acid (1 μg/ml) orvehicle control (1% Ethanol), and control experiments The cells wereincubated with solvent, 1 μg/ml Retinoic Acid, Blue Chamomile oil 1μg/ml+1% EtOH, Blue Chamomile oil 1 μg/ml+1 μg/ml Retinoic Acid, BlueChamomile oil 3 μg/ml+1% EtOH, Blue Chamomile oil 3 μg/ml+1 μg/mlRetinoic Acid, Blue Chamomile oil 10 μg/ml+1% EtOH, Blue Chamomile oil10 μg/ml+1 μg/ml Retinoic Acid (from left to right). As can be seen, acombination of Blue Chamomile oil 10 μg/ml+1 μg/ml Retinoic Acid showedthe best inhibitory effects on H69 cells.

FIG. 5: shows the median tumor volume of female C57BL/6 mice afterinjection of Pan02 tumor cells and treatment with Vehicle (Span20),MR31E25C1, Retinoic Acid or a combination of MR31E25C1 and RetinoicAcid. After 21 days, the groups treated with MR31E25C1 and incombination with Retinoic acid had reduced tumor volume in comparison tothe tumor-bearing untreated animals. The effect was however not observedin the group administrated only with Retinoic acid. As a matter of fact,treatment with Retinoic Acid alone even aggravated the situation incomparison to administration of Vehicle alone.

FIG. 6: shows the mean total metastases are for female C57BL/6 mice forthe kidney area, ovary area, peritoneum are, diaphragm area and spleenare after injection of Pan02 tumor cells and treatment with Vehicle(Span20), MR31E25C1, Retinoic Acid or a combination of MR31E25C1 andRetinoic Acid. The stacked bar graph reflects the cumulative metastasesarea measured in various organs per group. Animals treated withMR31E25C1 only or with Retinoic acid only resulted in only slightreduction of the overall metastasis development compared to theuntreated PAN02 inoculated group. Strikingly the MR31E25C1 incombination with the Retinoic Acid treatment led a marked regression ofmetastases in group 6.

TECHNOLOGICAL EXAMPLES OF EXTRACT AND TEST COMPOUND PREPARATIONS

The following Examples are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

Example 1 (Preparation of an Ethanolic Liquid Extract MR15E7)

200 g of tubular flowers from the inflorescence of Matricaria recutitaL. were extracted in a turbo mixer (3×1 min. at 8000 rpm, Eberbach®)with 1500 g ethanol absolute, corresponding to a drug solvent ratio of1/7.5 (w/w). The obtained mixture was stirred for one hour at atemperature of 40° C. The preparation was then filtrated twice over adeep layer cellulose filter (AF 15 Filtrox®).

1266 g of a clear brownish liquid extract with a solid content of 1.05%(m/m) were obtained.

Example 2 (Preparation of an Ethanolic Liquid Extract MR15E14)

200 g of tubular flowers from the inflorescence of Matricaria recutitaL. were extracted in a turbo mixer (3×1 min. at 8000 rpm, Eberbach®)with 1500 g ethanol absolute, corresponding to a drug solvent ratio of1/7.5 (w/w). The obtained mixture was stirred for one hour at atemperature of 40° C. The preparation was then filtrated twice over adeep layer cellulose filter (AF 15 Filtrox®).

1262 g of a clear brownish liquid extract with a solid content of 0.99%(m/m) were obtained.

Example 3 (Preparation of a Water-Free Liquid Extract MR15E7C1)

To 1266 g of an ethanolic liquid extract obtained (contains 13.3 gnative extract) in analogy to example 1 were added 26.6 g Span® 20(Sigma®)(sorbitan (mono)laurate) and 2.66 g Transcutol® HP (Gattefossé®)(diethylene glycol monoethyl ether), corresponding to a ratio of1/2/0.2. The ethanol was evaporated under reduced pressure (100 to 25mbar) and a bath temperature of 40° C. 45 g of a brownish liquid extractwith precipitate were obtained.

The 15.5 g native extract obtained was adjusted with 4.72 g Span® 20(Sigma®) and 0.47 g Transcutol® HP (Gattefossé®) in respect of the1/2/0.2 ratio. Also 100 ml of ethanol absolute were added to thismixture. The ethanol was evaporated under reduced pressure (80 to 25mbar) and a bath temperature of 40° C. 50 g of a brownish liquid extractwith precipitate were obtained.

Afterwards the extract was filtrated at 40° C. over a cellulose filter(TS 4 Filtrox®). 29 g of a brownish free-flowing liquid extract wereobtained.

The content of water was found to be 0.64% (w/w).

Example 4 (Preparation of an aqueous liquid extract CHARE) CHARE(AMT2003 Cam) was supplied by Auron Healthcare GmbH (Lot: 86118G001). Ithad a solid content of 11.68 mg/ml. The extract was prepared as follows:To 300.000 kg Aqua purificata 15.800 kg of tubular flowers from theinflorescence of Matricaria recutita L. were given under gentlestirring. The mixture was heated up to 90-95° C. in 30-35 minutes.Subsequent the mixture was cooled down to 30-35° C. and was thenfiltrated twice over a deep layer cellulose filter. 1% m/m of Mygliol812 N was added to the filtrate and gently stirred for 10 minutes.Subsequently the pH of the mixture was adapted to 6.3-6.7 with sodiumhydroxyde. Then the filtrate was micro-filtrated first through a 0.22 μmmembrane filter, the resulting filtrate through a 0.1 μm membrane filterand the resulting filtrate was subsequently filtrated through a 1000 kDaultra filter. A clear yellowish colored liquid extract was obtained and0.7% m/m Phenol puriss were added and the liquid mixed until the Phenolwas completely dissolved.

Example 5 (Preparation of Liquid Sample AMT(M))

AMT(M) is a mixture consisting of 4 components;

-   -   1) AMT2003 Vit(+): consists of 1.027 mg/ml of Dexpanthenol,        6.845 mg/ml Nicotinamide, 0.685 mg/ml Pyridoxin-HCL, 0.937        mg/mlRiboflavin Sodiumphosphate, 1.712 mg/ml Thiamine-HCL,        41.280 mg/ml Sodium ascorbate and additionally 1.79 mg/ml of        Chlorpheniramin maleate and was supplied by Auron Healthcare        GmbH (Ch.-B.:84105G005)    -   2) AMT2003Cal: consists of 0.5 g Calciumgluconate H₂O and 0.8756        g of calciumlactobionate 2H₂O and was supplied by Auron        Healthcare GmbH (Ch.-B.:0907000134)    -   3) AMT2003Ins: contains 100 IE/ml of human Insulin was supplied        by Auron Healthcare GmbH (Ch.-B.:0902000117)    -   4) MR15E14 (see example 2)

A stock solution of 10% AMT was prepared mixing 0.475 ml of AMT2003Vit(+)+0.320 ml of AMT2003Cal+0.025 ml of AMT2003Ins+1 ml of MR15E14(2.1 mg/ml)+8.18 ml of ultra pure water (UPW). Except of compound 4) thecomposition was prepared according to WO 03/101479 A1 for comparativestudies.

The final AMT concentration was 1% and 21 μg/ml for MR15E14.

Example 6 (Preparation of Liquid Sample AMT(CHARE))

AMT(CHARE) is a mixture consisting of 4 components;

-   -   1) AMT2003 Vit(+): consists of 1.027 mg/ml of Dexpanthenol,        6.845 mg/ml Nicotinamide, 0.685 mg/ml Pyridoxin-HCL, 0.937 mg/ml        Riboflavin Sodiumphosphate, 1.712 mg/ml Thiamine-HCL, 41.280        mg/ml Sodium ascorbate and additionally 1.79 mg/ml of        Chlorpheniramin maleate and was supplied by Auron Healthcare        GmbH (Ch.-B.:84105G005)    -   2) AMT2003Cal: consists of 0.5 g Calciumgluconate H₂O and 0.8756        g of calciumlactobionate 2H₂O and was supplied by Auron        Healthcare GmbH (Ch.-B.:0907000134)    -   3) AMT2003Ins: contains 100 IE/ml of human Insulin was supplied        by Auron Healthcare GmbH (Ch.-B.:0902000117)    -   4) CHARE (see example 4)

A stock solution of 10% AMT was prepared mixing 0.950 ml of AMT2003Vit(+)+0.640 ml of AMT2003Cal+0.050 ml of AMT2003Ins+0.36 ml of CHARE.The AMT composition was prepared according to WO 03/101479 A1 forcomparative studies.

The 100% AMT(CHARE) was further diluted to 10% AMT(CHARE) with UPW.

Example 7 (Preparation of Liquid Sample AMT(UPW))

AMT(UPW) is a mixture of consisting of 4 components;

-   -   1) AMT2003 Vit(+): has additionally 1.79 mg/ml of        Chlorpheniraminmaleate and was supplied by Auron Healthcare GmbH        (Ch.-B.:84105G005)    -   2) AMT2003Cal: consists of 0.5 g Calciumgluconate H₂O and 0.8756        g of calciumlactobionate 2H₂O and was supplied by Auron        Healthcare GmbH (Ch.-B.:0907000134)    -   3) AMT2003Ins: contains 100 IE/ml of human Insulin was supplied        by Auron Healthcare GmbH (Ch.-B.:0902000117)    -   4) Ultra pure water

A stock solution of 10% AMT was prepared mixing 0.950 ml of AMT2003Vit(+)+0.640 ml of AMT2003Cal+0.050 ml of AMT2003Ins+0.36 ml of UPW.Except of compound 4) the composition was prepared according to WO03/101479 A1 for comparative studies.

The 100% AMT(UPW) was further diluted to 10% AMT(UPW) with ultra purewater UPW.

Example 8 (Preparation of Sample Retinyl Acetate)

Retinyl Acetate (R7882, Lot: 029K12721) was purchased from Sigma Aldrich(Buchs, Switzerland). A stock solution of 1 mg/ml was prepared inethanol absolute. The final concentration used in the experiments was 1μg/ml in 1% ethanol.

Example 9 (Preparation of Liquid Sample Retinoic Acid)

all-trans-Retinoic acid (95152, Lot:1392337) was purchased from SigmaAldrich (Buchs, Switzerland). A stock solution of 1 mg/ml was preparedin ethanol absolute. The final concentration used in the experiments was1 μg/ml in 1% ethanol.

Pharmacological Examples in Cell Culture Example 10 (Effects of Samplesand Retinyl Acetate on A172 and U87's Cellular Growth)

The human glioblastoma cell lines A172 and U87 MG were cultivated inculture medium containing 10% FBS and kept in a humidified incubator at37° C. and 5% CO2. Cultures used in subsequent experiments were passagedless than 25 times. The cells were seeded at 100,000 cells in T25 cm2culture flasks (cell concentration of 10,000 cells/ml). MR15E7 andRetinyl Acetate prepared according to examples 1 and 8 respectively wereadded alone as individual or together in combination (A172 FIGS. 1A andU87 FIG. 1B). The solvent control was 2% ethanol.

Media were replaced once after 3 days of incubation with fresh media andsamples. After 6 days, all cells were harvested to be analyzed. The cellnumber was determined using a cell counter (Countess® automated cellcounter, Invitrogen).

Live cells were differentiated from dead cells by adding Trypan Blue.Live cells would be Trypan Blue negative since the dye would be excludedfrom viable cells.

The number of living cells obtained at the end of the experiment wastranslated into percent of cell count. Solvent control was set at 100%.The raw data (rd) of each well was then transformed to % cell countusing the formula: % cell count=(rd X/rd solvent control)×100. See FIGS.1A and 1B. The potential growth inhibitory effects of retinyl acetate (1μg/ml), MR15E7 (30 μg/ml) and the combination of 30 μg/ml of the extractand retinyl acetate (1 μg/ml) were observed on both the A172 (A) and U87MG (B) cell lines. The solvent control and the final ethanolconcentration for every well was 2%.

Results:

It appeared that retinyl acetate at 1 μg/ml do not affect cellulargrowth in A172 and U87 MG glioblastoma cells as indicated in FIGS. 1Aand 1B. The data derived from the experiments described in example 10,however, showed that MR15E7 reduced A172's cell numbers to 30% ascompared to solvent control (100%). The addition of 1 μg/ml of retinylacetate further reduced the cellular growth to 14%, demonstrating aclear efficacy enhancement of the combination compared to the compoundsalone (FIG. 1A). The effect is also seen similar in the U87 MG (FIG. 1B)but not as strong as observed for the A172.

Example 11 (Effects of Samples and Retinoic Acid on H460's CellularGrowth)

The human large cell lung cancer cell line, H460, was obtained from theAmerican Type Culture Collection. The cells were cultivated in RPMI-1640medium containing 10% FBS and kept in a humidified incubator at 37° C.and 5% CO₂. Cultures used in subsequent experiments were passaged lessthan 25 times. The H460 cells were seeded at 14375 cells/well (cellconcentration of 10,000 cells/ml) in 12 well plates and were left toadhere overnight.

MR15E14, CHARE and Retinoic acid prepared as described in examples 2, 4and 9 respectively including a solvent control (2% ethanol) were addedto the cells. Media were replaced once after 3 days of incubation withfresh media and samples. After 6 days, all cells were harvested to beanalyzed. The cell number was determined using flow cytometry. Livecells were differentiated from dead cells by adding Propidium iodide(PI). Live cells would be PI negative since the dye would be excludedfrom viable cells.

In short, cells were trypsinized, centrifuged and resuspended in 500 μlof media. 2 μl of PI (1 mg/ml) was added to 400 μl of cell suspension.Since the cells number present in 10 μl of the cell suspension wasmeasured, multiple measurements for each well were taken.

The cell numbers obtained at the end of the experiment was translatedinto percent cell count. Solvent control was set at 100%. The raw data(rd) of each well was then transformed to % cell count using theformula: % cell count=(rd X/rd solvent control)×100.

The results are depicted in FIG. 2. As can be seen, the effects ofretinoic acid (1 μg/ml), MR15E7 (21 μg/ml), CHARE (21 μg/ml) and thecombination of the extracts and retinoic acid (1 μg/ml) were observed onthe H460 cells. The solvent control and the final ethanol concentrationfor every well was 2%. As also shown in FIG. 2, 1 μg/ml of Retinoicacid, 21 μg/ml of MR15E14 and 21 μg/ml of CHARE slightly reduced thecellular growth of H460 when the cells were treated with theindividuals. However, when the same concentration of Retinoic acid wasgiven simultaneously with 21 μg/ml of MR15E14 or CHARE, theanti-proliferative effect was strongly enhanced.

Example 12 (Effects of AMT without Chamomile, with MR15E14 or withCHARE)

The following experiments were performed to compare the composition ofthe present invention with the teaching of WO 03101479 A1: The humanlarge cell lung cancer cell line, H460, was obtained from the AmericanType Culture Collection. The cells were cultivated in RPMI-1640 mediumcontaining 10% FBS and kept in a humidified incubator at 37° C. and 5%CO₂. Cultures used in subsequent experiments were passaged less than 25times. The H460 cells were seeded at 14375 cells/well (cellconcentration of 10,000 cells/ml) in 12 well plates and were left toadhere overnight. In one set of the experiment the cells weresubsequently incubated with MR15E14 (21 μg/ml), prepared according toexample 2, 1% AMT(M), prepared according to example 5 and 1% AMT(UPW),prepared according to example 7, in combination with retinoic acid (1μg/ml) or vehicle control (1% Ethanol) shown in FIG. 3A.

In another set of the experiment the cells were incubated with CHARE (21μg/ml), prepared according to example 4, 1% AMT(CHARE), preparedaccording example to 6 and 1% AMT(UPW), prepared according example 7 incombination with retinoic acid (1 μg/ml) or vehicle control (1% Ethanol)shown in FIG. 3B.

The H460 cells were incubated with the samples and sample pairs for atotal of 6 days with one dose and media refreshment. After 6 days, allcells were harvested to be analyzed. The cell number was determinedusing flow cytometry. Live cells were differentiated from dead cells byadding Propidium iodide (PI). In short, cells were trypsinized,centrifuged and resuspended in 500 μl of media. 2 μl of PI (1 mg/ml) wasadded to 400 μl of cell suspension. Since the cells number present in 10μl of the cell suspension was measured, multiple measurements for eachwell were taken. The cell numbers obtained at the end of the experimentwas translated into percent cell count. Solvent control was set at 100%.The raw data (rd) of each well was then transformed to % cell countusing the formula: % cell count=(rd X/rd solvent control)×100.

The results are illustrated in FIGS. 3A and 3B. As already earlier seenin FIG. 2, retinoic acid itself showed a growth retarding effect on H460large cell lung cancer cells and again the results indicate asynergistic effect between the chamomile extracts and retinoic acid inreducing cellular growth of H460. The addition of the AMT components tothe chamomile extracts did not enhance this effect (FIG. 4A) and, evenworse, addition of AMT components promoted cell proliferation in somecases (FIG. 4B). When the components of AMT were tested alone (withoutChamomile extracts) they markedly increased cell numbers by more than50% (FIGS. 3A and 3B). AMT containing the aqueous extract accordingexample 4 (CHARE) also caused an increase in cell growth. This was notseen with the ethanolic extract according example 2 (MR15E14), probablyas the antiproliferative activity is already alone somewhat strongerthen seen for the aqueous extract. Addition of retinoic acid abolishedthe growth promoting effect of AMT (UPW) and as also seen in theprevious pharmacological examples 10 and 11 enhanced theanti-proliferative effects of both chamomile extracts. The resultsdemonstrate the advantages of the use of a combination solely composedof chamomile and retinoic acid and the disadvantage of the complexmixture of compounds found in AMT.

Example 13 (Effects of Blue Chamomile Oil)

In this experiment the treatment of H69 cells with Blue Chamomile oil(according to Pharmacopeia: Matricaria aetheroleum) in combination withretinoic acid (1 μg/ml) or vehicle control (1% Ethanol), optionally inthe presence of Blue Chamomile, was carried out. For this experiment,the human small lung cancer cell carcinoma cell line H69 was firstsieved with a 40 μm filter to remove clumps before plating 50,000cells/well/800 μl (cell concentration of 63,500 cells/ml) in a 12 wellplate. Samples were added on the same day and the cells were incubatedfor a total of 7 days (Samples were: Solvent, 1 μg/ml Retinoic Acid,Blue Chamomile oil 1 μg/ml+1% EtOH, Blue Chamomile oil 1 μg/ml+1 μg/mlRetinoic Acid, Blue Chamomile oil 3 μg/ml+1% EtOH, Blue Chamomile oil 3μg/ml+1 μg/ml Retinoic Acid, Blue Chamomile oil 10 μg/ml+1% EtOH, BlueChamomile oil 10 μg/ml+1 μg/ml Retinoic Acid). After incubation, thecells were resuspended well before 100 μl of the suspension weretransferred to a 96 well plate. Duplicates were made. Equal volume ofCyQuant direct detection reagent was then added and incubated for 1 hrat 37° C. Only live cells would be stained. Fluorescence was measured at480/535 nm. The cell numbers obtained at the end of the experiment wastranslated into percent cell count. Solvent control was set at 100%. Theraw data (rd) of each well was then transformed to % cell count usingthe formula: % cell count=(rd X/rd solvent control)×100. The results canbe seen in FIG. 4 showing the treatment of H69 cells with Blue Chamomileoil in combination with retinoic acid (1 μg/ml) or vehicle control (1%Ethanol). As can be seen, a combination of Blue Chamomile oil 10 μg/ml+1μg/ml Retinoic Acid showed the best inhibitory effects on H69 cells.

CONCLUSIONS

As shown in the pharmacological examples 10-12 chamomile extractsprepared according to examples 1-4 are effective in inhibiting cancercell proliferation of different origins. As further shown in examples10-12, incubation of tumour cells with chamomile extracts together withretinoic acid or retinyl acetate enhanced this anti-tumour activitymarkedly and resulted at least in tumour cell death. While examples10-12 resulting only in a moderate and comparatively weakanti-proliferative effect in the case when the Vitamin A derivatives aregiven alone (the effect seems to be mainly mediated through thedifferentiating effects of the vitamins) it can be stated that acombination of chamomile extracts with Retinol or derivatives is in factacting synergistically leading to a pronounced increase in anti tumouractivity. It can be presumed further that the combination of the VitaminA/chamomile pair with further ingredients like those found in AMT israther contra productive and disadvantageous.

Example 14 (Preparation of a Water-Free Liquid Extract MR15E7C1)

To 350 g of a ethanolic liquid extract obtained (contains 49.21 g nativeextract) in analogy to example 1 were added 108.26 g Lutrol L44 (BASF,Art. Nr. 50143751, Batch.Nr. WPYF580B), corresponding to a ratio of1/2.2. The ethanol was evaporated under reduced pressure (100 to 25mbar) and a bath temperature of 40° C. to obtain a brownish liquidextract. The content of water was found to be 0.4% (w/w). Lutrol L44thus replaces in this example Span 20 used in example 3 of the presentapplication.

Example 15 (Efficacy Study in the Syngenic Pan02 Tumor Model in FemaleC57BL/6 Mice)

The purpose of this study was to obtain information about the efficacyof the oral combination of Chamomile extract (MR31E25C1 and Retinoicacid (RetAcid) in a syngenic orthotropic PAN02 tumor murine model.

Young (approximately 7 weeks) female C57BL/6 mice were purchased fromthe National Cancer Institute (Frederick, Md.) and maintained in AurigonLife Science GmbH's animal care facility with ad libitum access to waterand mouse chow (Ssniff® R/M-H). The animals were divided into groups of10 animals.

All animals, except those in the non-inoculated control group, weregiven an intrapancreatic injection of 2.5×105 Pan02 tumor cells each.Pan02 is a murine pancreatic adenocarcinoma cell line syngeneic to theC57BL/6 species. First treatment was given orally 15 days after theinoculation for a total period of 21 days.

Necropsy was performed at the end of the in-life phase or immediately onanimals found dead or killed in extremis. The weights of the brain,whole pancreas, pancreatic tumor, spleen, and liver were determined andthe number/size of metastases on the surface of the spleen and liver,peritoneal, kidney, ovary, diaphragm metastases, and metastases of thelymph nodes (if applicable) were recorded. Additionally, the size of thepancreatic tumors of the animals was measured.

The groups were as follows:

Group 1: Non-inoculated, untreated control group.

Group 2: Inoculated, untreated control group.

Group 3: Inoculated, vehicle Span® 20; 3× daily.

Group 4: Inoculated, MR31E25C1; 484.5 mg/kg, 3× daily.

Group 5: Inoculated, Retinoic Acid; 25 mg/kg 2× daily for first 2 days,10 mg/kg 1× daily for the rest of the treatment period.

Group 6: Inoculated, Retinoic Acid; 25 mg/kg 2× daily for first 2 days,10 mg/kg 1× daily for the rest of the treatment period and MR31E25C1;484.5 mg/kg, 3× daily.

After 21 days, the groups treated with MR31E25C1 and in combination withRetinoic acid had reduced tumor volume in comparison to thetumor-bearing untreated animals. The effect was however not observed inthe group administrated only with Retinoic acid. As a matter of fact,treatment with Retinoic Acid alone even aggravated the situation incomparison to administration of Vehicle alone. With respect to thecumulative metastases area measured in various organs per group, Animalstreated with MR31E25C1 only or with Retinoic acid only resulted in onlyslight reduction of the overall metastasis development compared to theuntreated PAN02 inoculated group. Strikingly the MR31E25C1 incombination with the Retinoic Acid treatment led to a marked regressionof metastases in group 6.

The invention claimed is:
 1. A method of treating a human suffering fromlung cancer or brain cancer consisting essentially of administering atherapeutically effective amount of an ethanol chamomile extract fromtubular flowers of Matricaria recutita and vitamin A to said humansuffering from said lung cancer or brain cancer to effectively treat thelung cancer or brain cancer in said human in need thereof.
 2. A methodaccording to claim 1, wherein the administering is via the respiratorytract, via oral administration, via injection, or via parenteralinjection of the human in need thereof.
 3. A method according to claim1, wherein the method further consists essentially of dimethylsulphoxide, diethylene glycol monoethyl ether, or a mixture thereof. 4.A method according to claim 1, wherein the amount of the ethanolchamomile extract from tubular flowers of Matricaria recutita is about50 mg to about 1500 mg and the amount of the Vitamin A is about 3 mg toabout 90 mg.